The present invention relates to a method of competitively excluding pathogens capable of intestinal colonization, especially Salmonella, from the digestive tract of a bird, comprising administration of a competitive exclusion (CE) culture essentially free from abundant gas forming bacteria in ovo to a fertile bird egg.
Poultry is the most important source of human gastrointestinal infections, such as Salmonella and Cambylobacter infections. For a long time Salmonella infantis has been a very common Salmonella type that cause human Salmonella infections. Now there has also been an alarming increase in the incidence of human food poisoning associated with Salmonella enteritis PT4. See L. Nuotio, C. Schneitz, U. Halonen and E. Nurmi, British Poultry Science 33, 775-779 (1992). Much of the increase has been associated with eating raw or undercooked eggs that were contaminated with salmonellas, but broilers are also a considerable reservoir of infection for man, even in relation to handling of newly-hatched chicks. Broiler chicks can be infected through transovarian transmission and the salmonellas may also spread via contaminated feed. There are several reports in which the epidemically implicated or suspected vehicle of cambylobacteriosis has been raw, barbecued or undercooked chicken. In addition to human infections the broilers themselves may be infected by pathogens causing mortality, such as some Clostridium species.
Competitive exclusion is a method of preventing pathogenic bacteria from colonizing birds and thus infecting man and birds themselves. Because of the diverse sources of contamination it is difficult to administer a competitive exclusion culture to a bird before it is colonized by pathogenic bacteria. N. A. Cox et al., Poultry Science 71, 1781-1784 (1992), describes a study of in ovo administration of a competitive exclusion culture treatment to broiler embryos. U.S. Pat. No. 5,206,015 discloses a method and apparatus for introducing probiotic bacteria into the digestive tract of a bird in order to exclude undesirable bacteria therefrom, and inoculated eggs produced thereby. In a preferred embodiment of the invention, a fertile bird egg is administered a Salmonella competitive exclusion culture, such as disclosed in U.S. Pat. No. 4,689,226 and U.S. Pat. No. 4,335,107. The culture may optionally include an oxygen scavenging agent such as cysteine as described in U.S. Pat. No. 4,657,762. The culture preferably comprises at least one anaerobic bacteria of intestinal origin.
According to U.S. Pat. No. 5,206,015 the culture is preferably administered into the amnion or the air cell of the egg. Most preferably the bacterial culture is deposited in the air cell. However the amnion of the egg for the bacterial deposition seems to be a problem. In fact, according to the description, putting the CE culture into the amnion killed most of the chicks before hatch, even at 1:1,000,000 dilution.
The above mentioned patent discloses also the problem that when an undiluted CE culture was placed in the air cell, hatchability was significantly reduced when compared to controls.
The object of the present invention is to provide a better and more advantageous method compared to prior art for competitively excluding pathogens capable of intestinal colonization, from the digestive tract of a bird. The pathogens capable of intestinal colonization are e.g. Salmonella sp., Cambylobacter sp. and Escherichia Coli. 
Specifically the present invention provides an advantageous method for competively excluding Salmonella from the digestive tract of a bird prior to colonization by Salmonella.
Competitive exclusion essentially comprises the prevention of the population of the gut by pathogens through pre-populating the gut with non-pathogenic microflora. Typically the bacteria strains in a CE culture produced by the method described in U.S. Pat. No. 5,206,015 include spore-forming bacteria, such as different Clostridium species, (see H. Pivnick, B. Blanchfield and J. Y. D""Aoust, Journal of Food Protection 44, 909 (1981) and H. Pivnick and E. Nurmi, Developments in Food Microbiology 1, 41 (1982)), which are known to form abundantly gas.
It has unexpectedly been found that the hatchability is significantly increased when CE cultures, which are essentially free from abundant gas forming bacteria, are administered in ovo to a fertile bird egg, either to the region of the air cell or the amnion, compared with that described in U.S. Pat. No. 5,206,015. Especially the difference between the hatchabilities of the eggs where the CE cultures are administered into the amnion is drastic showing that CE cultures which are essentially free from abundant gas forming bacteria can be administered to the amnion without killing the embryo.
Administration of the CE culture preparations to the amnion is more beneficial compared to the air cell, because the bird comes in contact with the protective bacteria in the earliest possible stage. Also the anaerobic bacteria in a CE culture preparation are more viable in the amnion than in the air cell. Thus the problem with e.g. Salmonella infected breeders and Salmonella contaminated egg shells will be minimized.
Moreover it has been found that CE cultures may be coadministered in ovo with commonly used vaccines, such as Marek""s vaccine, in the poultry industry. The vaccines are usually administered to the amnion, which makes the region of amnion more preferable administration site than the air cell.
The invention provides an improved method for competitively excluding pathogens capable of intestinal colonization, such as Salmonella or Cambylobacter, comprising administration in ovo to a fertile bird egg of a CE culture essentially free from bacteria which abundantly form gas. The CE culture may be any CE culture essentially free from abundant gas forming bacteria derived from digestive tract of mature birds which are free from pathogens, such as Salmonella. It may be undiluted or diluted. Preferably the CE culture is prepared as described in U.S. Pat. No. 4,689,226 or EP-A-479 820 which are checked to be essentially free from abundant gas formers,such as Clostridia.
U.S. Pat. No. 4,689,226 describes a bacterial preparation for the prophylaxis of intestinal disturbances in poultry caused by pathogenic bacteria. The preparation is made by anaerobically cultivating either separately or together bacteria strains of normal alimentary tract bacterial species having an adhesion efficiency onto the epithelial cells of the alimentary tract of poultry of at least 10 bacteria per epithelial cell, and isolating the cultivated bacteria. The bacterial strains are cultivated preferably together with epithelial cells from the alimentary tract, for example from the crop of the chicken. After the cultivation, the bacteria are isolated from the culture broth and finished to a preparation, for instance by lyophilization. The test for adhesion may be carried out by a known method, for instance according to the Fuller adhesion test.
According to U.S. Pat. No. 4,689,226 the isolation of suitable bacteria strains can be performed as follows:
The content of the alimentary tract of an adult chicken is mechanically removed. After this, loose and weakly attached bacteria are removed, for instance by washing with a phosphate buffered saline. The washed alimentary tract, or part of it, preferably the crop or the caecum with the attached bacteria remaining, is minced, suitably diluted and cultured to obtain pure cultures. Bacterial strains with good adhering ability are selected for the cultivation of the final preparation. Especially good results are obtained when two or more bacterial strains are cultivated together.
EP-A-479820 describes a bacterial preparation useful for the prophylaxis of colonization of human pathogenic bacteria, especially Cambylobacter species, in poultry comprising bacteria derived from an adult bird, from the microecological niche which pathogenic bacteria occupy. The bacterial flora of the preparation is derived from the caecal mucous layer of adult birds. Highly effective floras are those which are obtained by culturing the bacterial flora from the mucous layer of the caecal wall from an adult bird under either anaerobic or microaerophilic conditions, preferably in a mucin broth.
The culture may optionally include an oxygen scavenging agent such as cysteine as described in U.S. Pat. No. 4,657,762. Most preferably the CE culture is prepared as described in U.S. Pat. No. 4,689,226. One of the most preferable CE cultures is commercially available from Orion Corporation marketed under the trademark BROILACT(copyright), which is cultured from biological material deposited under the name xe2x80x9cProduction inoculum SHGO1Bxe2x80x9d and under ATCC accession number 55715. The biological material was deposited on Oct. 3, 1995, with the American Type Culture Collection, Va. 20110-2209 U.S.A.
The CE culture is administered either into the air cell or the amnion of a fertile bird egg close to hatch, for example, when at least 60%, preferably 70%, more preferably 75% of the incubation time has elapsed. The CE culture is administered in an amount effective to colonize the digestive tract of the embryonic bird. Preferably the CE culture is administered into the amnion including the amniotic fluid, the yolk sac and the embryo itself. A suitable dose for BROILACT(copyright) is from about 0.01 mg to 1 mg per egg, preferably about 0.1 mg per egg when administered into the amnion. After injection the eggs are incubated to hatch. The digestive tracts of the hatchlings of these eggs are colonized by the CE culture at the time of hatch.
The present invention may be practiced with any type of bird egg, including chicken, turkey, duck, goose, quail or pheasant eggs. Preferred eggs are chicken and turkey eggs. Most preferred eggs are chicken eggs.
Chicken eggs are injected on about the fifteenth to nineteenth day of incubation, preferably on the nineteenth day of incubation.
The injection of a bird egg may be done manually, but an automated injection is preferred. A suitable apparatus is described in U.S. Pat. No. 5,206,015.